Engineering placenta‐like organoids containing endogenous vascular cells from human‐induced pluripotent stem cells

Abstract The placenta is an essential organ that maintains the health of both the fetus and its mother. Understanding the development of human placenta has been hindered by the limitations of existing animal models and monolayer cell cultures. Models that can recapitulate the essential aspects of human placental multicellular components and vasculature are still lacking. Herein, we presented a new strategy to establish placenta‐like organoids with vascular‐like structures from human‐induced pluripotent stem cells in a defined three‐dimensional (3D) culture system. The resulting placenta‐like tissue resembles first‐trimester human placental development in terms of complex placental components and secretory function. The multicellular tissue was characterized by the inclusion of trophoblasts (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and other endogenous vascular cells), which were identified by immunofluorescence, flow cytometry analyses, real‐time quantitative reverse transcription polymerase chain reaction and single‐cell RNA‐seq. Moreover, the 3D tissue was able to secrete the placenta‐specific hormone human chorionic gonadotropin β (hCG‐β) and vascular endothelial growth factor A (VEGFA). The tissue responded to the inflammatory factor tumor necrosis factor‐α (TNF‐α) and VEGF receptor inhibitors. This new model system can represent the major features of placental cellular components, and function, which have not been realized in 2D monolayer cultures. The developed tissue system might open new avenues for studying normal early human placental development and its disease states.


| INTRODUCTION
The placenta is the first and largest fetal organ to develop and crucial for the health of both the fetus and its mother. It has been recognized as having a lifelong impact on their long-term well-being. 1 As a highly specialized extraembryonic organ, the placenta forms a maternal-fetal interface that allows for efficiently transferring nutrients and oxygen from the mother to the fetus. A successful pregnancy requires rapid growth and increased blood flow of the placenta to support the steadily increasing metabolic demands and oxygen of the growing fetus. 2 The coordinated development of the highly vascularized placental villous tree is necessary for continued fetal growth and wellbeing. Dysfunction in the placental vasculature leads to changes in the function and development of specialized placental cells called trophoblasts [3][4][5] as well as common placental disorders such as preeclampsia and fetal growth restriction. 6 Pre-eclampsia is characterized by reduced placental vascular perfusion and an altered angiogenic response, leading to maternal-fetal morbidity. 7 Various cell lines and animal model systems have been established to study placental biology and its associated diseases. Although much of our knowledge about trophoblast lineages and placental development comes from animal models, mammals (e.g., mice and primates) display significant diversity in placental physiology, particularly in terms of degrees of trophoblast invasion into the uterine tissue and the formation of cell layers between the fetal and maternal circulation. 8 No equivalent animal system can accurately represent human placental development. Several trophoblast cell lines, primary cells, and villous explants have been utilized as in vitro models in placental research. 1,9,10 However, the cell lines (e.g., BeWo, JEG-3) are usually generated from a variety of sources, such as primary placental tissue and malignant tissue. Each cell line has a different phenotype and cannot represent the multiple trophoblast subtypes and physiologically relevant features of in vivo human placental tissue. Although human primary trophoblast cells and villous explants are useful for investigating physiological and pathological conditions, such as embryonic development, fetal disorders, and immune diseases, 11,12 these cells are quite difficult to obtain due to the limited source of primary human tissue.
Advances in stem cell biology have facilitated the development of a new in vitro system to model human organ development and disease. 13 In vivo, trophoblast cells perform the major functions of the placenta. 14 After exposing to BMP4 in a two-dimensional (2D) monolayer culture, human pluripotent stem cells (hPSCs) can be successfully differentiated into trophoblast cells, the major components of the placenta. [15][16][17] In addition, human trophoblast stem cells (hTSCs) derived from first-trimester placenta and the trophectoderm exhibit the main characteristics of first-trimester trophoblasts. 18 However, the hTSC lines grow as monolayers and thus do not reflect the complex morphological complexity of the early placental villi and vasculature.
Due to the self-renewal and self-organized property of stem cell, organoids with 3D structure are generated in recently years. 13 Stem cell-derived organoids can replicate the near-physiological structure and function of native human organs, such as the intestine, retina, kidney, liver, and brain. 19 Compared with the traditional 2D cell cultures, 3D organoids are more physiologically relevant to primary tissues in terms of cell components, architectures, and functions. They offer a pivotal system to study cellular models of human tissue and disease. 20 Trophoblast organoids derived from first-trimester placenta were recently shown to provide a near-physiological model by resembling the villous placenta in vivo, and this model was used to study the maternal-fetal interactions that occur during human placentation. 21 In addition, pluripotent stem cell-derived trophoblast organoids provide an alternative way to study the human placenta development and disease. [22][23][24] However, human placenta is a complex organ with vasculature; it contains not only the trophoblasts but also other placental components, such as vascular cells. Models that can recapture the complex cellular components, vascular-like structure, and functional aspects of the human placenta are still scarce.
With the aim to generate the vascularized trophoblast organoids, we propose a new strategy to engineer placenta-like organoids that contain endogenous vascular cells from human-induced pluripotent stem cells (hiPSCs) in a defined 3D culture system in this study ( Figure 1). The developed placental tissue contained trophoblast lineages, vascular lineages, and a placental villous-like structure, resembling the key features of first-trimester human placenta in terms of cellular components, and secretor function. Single-cell RNA-seq (scRNA-seq) was used to reveal the coordinated differentiation of trophoblasts and vascular cells from hiPSCs in the 3D culture system. Such development has not been reported in 2D cultures. This 3D model system might provide a new platform for studying normal early human placental development and its disease states.

| Differentiation of placenta-like tissue from hiPSCs in a 3D culture
In vivo placental development occurs in a dynamic and complex 3D microenvironment. To achieve the conditions suited for generating placenta-like tissue from hiPSCs, we established a multistep protocol to simultaneously induce trophoblast lineage differentiation and vascular lineage specification in a 3D culture system. BMP4 facilitates the induction of trophoblast and mesoderm lineages from hiPSCs and human embryonic stem cells in monolayer cultures. 25,26 A natural extracellular matrix (Matrigel) can contribute to the formation of 3D tissue and even organoids. 27,28 We optimized the culture conditions and investigated the feasibility of differentiating hiPSCs into placentalike tissues using BMP4 induction in 3D Matrigel under low oxygen conditions. The hiPSCs were seeded onto a uniformly structured micropillar chip to facilitate the generation of an array of 3D aggregates. 29 The cellular aggregates were then treated with BMP4 Flow cytometry analysis showed that the 3D clusters in the Matrigel culture exhibited higher viability compared with those cultured without Matrigel ( Figure S1). Because the complicated components of the culture medium can affect the differentiation of 3D cultures, we further optimized the culture medium conditions as shown in Figure S2.
Under the optimized conditions, we examined the trophoblastspecific cell types in the 3D clusters using immunofluorescence analysis. The results revealed that the 3D cultures contained CDX2 + CTBs

| Single-cell transcriptome atlas of placentalike tissues derived from hiPSCs
To comprehensively investigate the differentiation lineages of the placenta-like tissue, the samples underwent scRNA-seq, which was performed by 10x Genomics. We obtained 6507 and 9804 highquality scRNA-seq profiles from the samples after 9 and 24 days of F I G U R E 1 Schematic of early human placental development in vivo and in vitro. (a) Early human placental development in vivo. During pregnancy, the oocyte combines with sperm to form the zygote, thereby triggering embryogenesis. After fertilization, the blastocyst segregates into two lineages, the trophectoderm (TE), and the inner cell mass (ICM). The TE gives rise to the epithelial portion of the human placenta. As the main component of human placenta, the trophoblast is composed of three subtypes: CTBs, STBs, and EVTs. The multinucleated STBs line the outermost surface of the human placenta and subsequently form the major cellular barrier between the feus and mother. The EVTs invade into the decidua and remodel the maternal blood supply. (b) Illustration of human placental model generation in vitro. hiPSCs were seeded onto micropillar chips and treated with BMP4 to generate 3D clusters with trophoblast and mesodermal lineages under 3D culture conditions. The 3D clusters gradually grew into millimeter-sized tissues when treated with a cohort of factors (e.g., VEGFA, bFGF, and R-spondin 1). The formed 3D tissue contained trophoblast subtypes (CTBs, STBs, and EVTs) and vascular cells. (c) Characterization of hiPSC-derived placenta-like 3D tissue through immunofluorescence staining, qRT-PCR, single-cell RNA-seq, and flow cytometry. differentiation (D9 and D24), respectively. After rigorous filtering, 12 transcriptionally distinct clusters were generated by unsupervised clustering of the entire pooled dataset of 16,311 cells. These clusters were illustrated by uniform manifold approximation and projection (UMAP) using the Seurat clustering method (Figure 3a). Differential gene expression analysis was guided by established placenta-specific markers [30][31][32][33] and indicated that these cell clusters were CTBs, STBs, EVTs, proliferating cells, and vascular cells (Figure 3a). The violin plots illustrate the expression of established cell-specific markers across different clusters (Figures 3b-f).
According to published databases, 27  The time points of the differentiation process match well with the pseudotimes ( Figure S3b). We then examined the top 50 differentially expressed genes (DEGs) in the bifurcation of the first branchpoint.
The results indicated that these genes enriched in this branchpoint were tightly related with placental trophoblast development, such as KRT8, KRT18, and CD9 ( Figure S3d). It revealed that the trophoblast lineages within the 3D cultures seemed to share common origins during the process of placenta development.
In addition, the placenta-like organoids at 9 and 24 days of differentiation were also individually analyzed as shown in Figures

| Functional characterization of placenta-like tissue derived from hiPSCs
The in vivo placenta secretes a series of hormones and growth factors to maintain placental development and fetal growth during pregnancy.
Human chorionic gonadotropin-β (hCG-β) is a hormone that is mainly produced by STBs and serves as a critical marker for pregnancy tests. 37,38 Vascular endothelial growth factor A (VEGFA) is an important signal protein in regulating the early formation of blood vessels in human placenta. 39 We identified hCG-β and VEGFA in the medium of the 3D cultures by enzyme-linked immunosorbent assay, indicating the secretory ability of the produced placenta-like organoids in vitro (Figures 6a,b). To determine the response of the placenta-like organoids to inflammatory factors, the 3D cultures were exposed to tumor necrosis factor-α (TNF-α). Increased expression of the adhesion protein ICAM-1 was observed after TNF-α stimulation (Figure 6d,e), reflecting the feasibility of this placental model to recapitulate the humanrelevant responses to an inflammatory stimulus. After treatment with VEGF receptor inhibitors, which are known to disrupt VEGFA signaling, the vascular-like network was compromised as indicated by decreased CD31 and PDGFβ expression (Figure 6f,h). These results demonstrated the mature function of the vascular cells within the 3D tissue, which could respond to external stimuli.

| DISCUSSION AND PERSPECTIVE
In this study, we established a defined protocol to engineer hiPSCderived placenta-like tissue containing endogenous vascular cells in a defined 3D culture system. The generated placental organoids were able to recapitulate the multicellular components and functional features of first-trimester human placental tissue. By exposing the culture to a cohort of chemical factors, the hiPSCs could self-organize into 3D tissues with trophoblast lineages (CTBs, STBs, and EVTs) and vascular lineages in the 3D culture, as identified by scRNA-seq, qRT-PCR, immunofluorescence, and flow cytometry analysis. In particular, we identified endogenous vascular cells, such as endothelial cells and pericytes, that have not been observed in 2D cultures. Moreover, the 3D tissue could secrete both the placental-specific hormone hCG-β and blood vessel-related VEGFA.
These capabilities reflect the physiologically relevant functional aspects of this human placenta-like organoid.
In vivo, a complex vascular network is required to ensure sufficient oxygen and nutrient transfer from the placenta to the fetus during pregnancy. In this study, we established a 3D human placen- Trophoblast organoids derived from first-trimester placenta and hPSCs provide a near-physiological model because they resemble the villous placenta in vivo. This model has enabled the study of maternal-fetal interactions that occur during human placentation. [21][22][23][24]41 However, the human placenta is a complex vascularized organ that contains the trophoblast as well as other placental components, such as vascular cells. In this study, we made the first attempt to create a human placental model from hiPSCs with a vascular-like network that included both the trophoblast and vascular lineages. This 3D model system has the potential to be used to study interactions between trophoblasts and other placental cells and to explore the maternal-fetal interface of early pregnancy. It might also be used to probe gestational diseases associated with impaired vascular networks, such as fetal growth restriction and pre-eclampsia. In addition, it can serve as a potential platform for drug testing and for studying pathogen infection during pregnancy.
Considering that Matrigel hydrogel matrix were used to generate organoids such as brain, kidney, and liver, we establish placenta-like organoids in this study using Matrigel. Due to Matrigel suffering from high batch-to-batch variability, we will aim to develop a more con-

| hiPSCs culture
hiPSCs generated from reprogramed skin fibroblasts were maintained under feeder-free conditions in accordance with our previous study. 29 Two hiPSC lines were cultured on Matrigel-coated plates in mTeSR1 medium and passaged at the ratio of 1:6 to 1:5 with Accutase every 4-5 days.

| Formation of hiPSC-derived placenta-like endogenous vascular cells
To generate placenta-like organoids, hiPSCs were digested into small pellets (containing several cells) using Accutase and resuspended in Knockout Serum Replacement (KSR) medium supplemented with DMEM: F12 medium, 20% KOSR (Knockout Serum Replacement), 1X GlutaMax, 1X nonessential amino acid (NEAA), and 1X penicillinstreptomycin. Next, 5 Â 10 6 cells were seeded onto a micropillar chip for cell aggregation as previously described 43     penicillin-streptomycin with 50 ng/ml FGF2, 50 ng/ml VEGFA, and 5 μM Y-27632). All media were changed every 2-3 days. The 3D cultures were either directly analyzed or extracted from the gels for subsequent analysis. The placenta-like tissue could be cultured for more than 1 month.

| Cryopreservation ang immunofluorescence staining
The tissue cryopreservation and immunofluorescence staining procedures are described in detail in previous study. 43 In brief, 3D cultures harvested at different days were fixed with 4% paraformaldehyde for approximately 30 min at room temperature. They were then washed three times with PBS and transferred to 30% sucrose overnight for dehydration. On Day 2, the placenta-like tissues were embedded in optimal cutting temperature compound (Sakura) and then cryosectioned into 15-20 μm sections using a Leica CM1950 cryostat. Before conducting the immunofluorescence staining, we washed the cryosectioned slices with PBS and then permeabilized the frozen sections with Triton X-100 (0.2%), followed by blocking with 10% goat serum (ZSGB-Bio) for 1 h at 37 C. After blocking, the sections were washed with PBS and then incubated with primary antibodies overnight at 4 C, followed by staining with secondary antibodies for 1 h at 37 C.
The antibodies used are described in detail in Table S2. The sample images were captured using a confocal microscope (Olympus) after counterstaining the nuclei with DAPI.

| Enzyme-linked immunosorbent assay
The levels of VEGFA and hCG-β secreted by the established placentalike tissue were detected using human VEGFA (DY293B; R&D) and hCGβ ELISA kits (ab100533; Abcam) according to the manufacturer's instructions. After culturing for 48 h, the collected culture medium was centrifuged to remove debris. Then the supernatant was stored at À80 C before use. The absorbances were measured using TECAN Infinite M Nano.

| Flow cytometry
The Matrigel-embedded clusters were dissociated from the matrix using Cell Recovery Solution (354253; Corning). Organoids were dissociated by incubating in trypsin (0.25%) at 37 C for about 10 min, followed by incubation at 4 C for 20 min to depolymerize the Matrigel. Following incubation, the cells were washed with medium containing FBS, and then 40-μm cell strainers (2340; Falcon) were used to obtain single cells. The cells and isotype-matched controls were stained with ITGA2-PE, KDR, CD140 (PDGFRβ), and CD31 for analysis (Table S2). The viability of each  Images of the TEM sample grids were captured with a Spirit transmission electron microscope (FEI) operated at 100 kV.

| RNA extraction and real-time reverse transcription PCR
The RNA extraction and PCR procedures are described in detail in previous study. 43 In brief, total mRNA from the 3D tissue or hiPSCs were extracted using Trizol reagent. The RNA concentration and quality were measured using NanoPhotometer (Implen). cDNA was generated and then amplified using Ex Taq DNA polymerase (Takara). The amplification conditions were listed as following: 1 min of denaturing at 94 C, 45 s of annealing at 58 C, and 30 s of extension at 72 C, with 40 cycles.  Data are presented as the mean ± SEM. The data were analyzed using Student's t-test (*p < 0.05) 4.9 | scRNA-seq data processing Seurat (version 3.0) was used to conduct quality control and filtering.
The quality control parameters to retain cells were defined as a mitochondrial gene content of less than 10%. The quality control and filtering results are shown in violin plots ( Figure S6). After rigorous quality control and filtering, 6744 single cells from Days 9 and 10, 259 single cells from Day 24 remained. Unsupervised lineage trajectory analysis on a down-sampled set of Days 9 and 24 cells was then performed using Monocle 2 (version 2.10.0). Briefly, the genes used to order cells along the trajectory were the genes whose mean expression R value was 0.05. The branched expression analysis modeling function of Monocle 2 was used to identify DEGs (significantly DEGs) (q value % 1eÀ4) at branch points along the trajectory. In addition, a branched heatmap was used to visualize significant branch-specific DEGs to detect changes in both cell fates concurrently. Hierarchical clustering of branch-specific DEGs was used to identify genes with similar lineage-dependent expression patterns, which were then classified into upregulated or downregulated branch-specific DEGs.

| Statistics and reproducibility
All experiments described in this work were repeated, and similar results were obtained from independent samples. Data are presented as the mean ± SEM. For statistical significance, *p < 0.05 was accepted as statistically significant. At least 15 formed tissue samples in each group were randomly investigated by qRT-PCR, immunohistochemical staining, and quantitative analysis.

CONFLICT OF INTERESTS
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The raw scRNA-seq data files of this work have been deposited and available in the National Center for Biotechnology Information Sequence Read Archive repository (https://dataview.ncbi.nlm.nih. gov/object/PRJNA694235?reviewer=dvcl9ohi7i8g0ehh3amh9ef001) with accession number PRJNA694235. Other data supporting the results of this work can be obtained from the corresponding author.